The capacity for regeneration of neuronal and non-neuronal populations in the olfactory epithelium (OE) is well-known and extends throughout life. By virtue of their accessibility, the neurocompetent stem and progenitor cells of the OE are attractive candidates for use in autologous cellular therapies. The regulatory signals and mechanisms that govern these processes are not well understood. An in-depth analysis of the regulation of olfactory epitheliopoeisis is needed in order to exploit the stem and progenitor cells fully, but progress has been slow. We have developed a tissue culture assay in which cells from the neonatal or adult lesioned OE grow within spheres that form at the air-media interface of culture well inserts in a condition- dependent manner. Remarkably, cells that are cultivated in 3-dimensions (3-D) retain the potency and plasticity to participate in the regeneration of the OE in the animal, which stands in sharp contrast to the failure of cells grown in 2-D to do so. Two Aims are proposed to explore the cues regulating cell generation and differentiation taking advantage of the in vivo-like properties of the sphere assay. Aim 1 will test how growth factors, alone or in combination, will affect the growth of OE cells in the 3-D cultures. Some of the factors have a previously established role in the turnover and regeneration process in the OE; they will calibrate the assay, providing registration of the in vitro and in vivo results. For the majority, little is known of their role in the OE, although they function elsewhere in cell birth or differentiation. In contrast, Aim 2 will determine the relevant cues within a complex molecular mix that is known to play an important role in regulating the OE. In this case, culture media conditioned by the growth of a lamina propria cell line (derived from the part of the mucosa that is deep to the OE) is required for the formation of spheres from adult lesioned OE on inserts. We will take both a candidate molecule and an open-ended approach to identifying the relevant factors. PUBLIC HEALTH RELEVANCE: The health relevance of the work resides in the ultimate goal of achieving regulated controlled growth of olfactory stem cells and progenitor cells. Besides the goal of helping patients with dysosmias or anosmias, the neurocompetent tissue stem cells may prove useful for repairing other parts of the nervous system, including the spinal cord after it is injured.